The present invention relates to a novel enzyme. In particular, the invention relates to a novel cyclophilin-like peptidyl prolyl cis-trans isomerase.
In most protein over-production strategies, strong promoters capable of directing very high levels of transcription are used to over-express genes encoding heterologous and homologous proteins. Limitations in protein secretion are likely to be due to bottlenecks at the translational and post-translational levels (Tsuchiya, K. et al., (1992) Applied Microbiology and Biotechnology 38:109-114). Proper folding of the protein is required for export competence. Overexpressed heterologous proteins may fold improperly and then be degraded during protein traffic through the secretory pathway. Therefore, the correction of folding defects is desirable in order to increase protein secretion.
The folding of a protein is catalysed by an number of factors, including two isomerase families, namely protein disulphide isomerase, catalysing disulphide bond formation, and peptidyl prolyl cis-trans isomerase, catalysing the isomerisation of Xaa-Proline bonds.
Overexpression of protein disulphide isomerase in S. cerevisiae results in an increase in secretion of human platelet-derived growth factor (Robinson et al., 1994). However, the overproduction of A. niger protein disulphide isomerase did not result in an increase in secretion of hen egg white lysozyme (HEWL) or glucoamylase (Ngiam C., Jeenes, D. J. J., Punt, P. J., van den Hondel, C. A. M. J. J., Archer, D. A. (1998); Characterisation of a foldase, PDIA, in the protein secretory pathway of Aspergillus niger, submitted.)
One of the slowest steps in protein folding is the cis-trans isomerisation of Xaa-proline bonds. This isomerisation is markedly accelerated when peptidyl prolyl cis-trans isomerases are present. Peptidyl prolyl cis-trans isomerases of the cyclophilin family from different organisms have been shown to possess foldase activity in vitro (Schxc3x6nbrunner E. R., Mayer S., Tropschug M., Fischer G., Takahashi N., Schmid, F. (1991); Catalysis of protein folding by cyclophilins from different species. J Biol Chem 266: 3630-3635). These isomerases are inhibited by the immuno-suppressant drug cyclosporin A. The effects on secretion of beterologous peptides by overexpression of peptidyl prolyl cis-trans isomerases are however not known in the prior art.
Proteins active in the E.R. are targeted to this compartment by a carboxy terminal extension of 4 amino acids. In A. niger HDEL (SEQ ID NO: 7); and KDEL (SEQ ID NO: 9) have been reported to function as an E.R. retention signal (Jeenes D..J., et al., (1997) Gene 193:151-156).
In a first aspect of the present invention, there is provided a method for producing a secretable polypeptide in an host cell, comprising overexpressing a peptidyl prolyl cis-trans isomerase in the cell, thereby increasing the yield of the secreted polypeptide.
In a second aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, having a signal sequence at the N-terminus and an endoplasmic reticulum retention signal at the C-terminus, and a molecular weight of 20.7 kDa and a deduced isoelectric point of 6.27.
In a third aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, encoded by a nucleic acid capable of hybridising under conditions of low, medium or high stringency with a 17 base oligonucleotide derived from SEQ ID No. 1.
In a fourth aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, encoded by a nucleic acid capable of hybridising under conditions of low, medium or high stringency with a 20 base oligonucleotide derived from SEQ ID No. 2.
In a fifth aspect, the invention relates to a polypeptide possessing foldase activity characterised by having a capability to catalyse the cis-trans isomerisation of a peptide bond on the N terminal side of proline residues in polypeptides, which is at least 40% homologous to SEQ. ID. No. 2.